US 8,796,037 B2Granted

Method of detection, separation and identification for expressed trace protein/peptide

OwnerKazuhiro Imaiinventor

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Abstract

A method of detection separation and identification for expressed trace protein/peptide; and a system therefor. There is provided a method of detecting, separating and identifying a minute amount of expressed protein and/or peptide, characterized in that a fluorescent derivative of protein and/or peptide contained in a test subject sample having been labeled with a fluorescence reagent is applied to HPLC; a fluorescent fraction is collected and subjected to enzymatic hydrolysis; mass-spectrometry of the resultant fluorescence-labeled fragments and non-fluorescence-labeled fragments is carried out; and the thus obtained ion molecular weight information on each of the fragments is collated with an available protein and/or peptide fragment database to thereby accomplish a structural analysis. Further, there is provided an identification system therefor.

Claims11

Dependent claims

A method for detecting, separating and identifying an expressed trace protein and/or peptide in a test sample, comprising:

converting a protein and/or peptide in a test sample to a fluorescent derivative by labeling said protein and/or peptide with a fluorescent derivatization reagent, wherein said fluorescent derivatization reagent, which does not fluoresce itself, is one selected from the group consisting of DAABD-X, TAABD-X, DAABSeD-X, TAABSeD-X, DAABThD-X, and TAABThD-X, wherein X represents Cl or F
subjecting the labeled protein and/or peptide to one-dimensional or two-dimensional HPLC/fluorescence detection, to obtain fluorescent fractions,
applying the fluorescent fractions to enzymatic hydrolysis to obtain digested peptide fragments,
subjecting the digested peptide fragments to second stage HPLC/fluorescence detection to obtain a fluorescent chromatogram,
applying peptide fractions of the fluorescent chromatogram to mass spectrometry or MS/MS analysis,
collating the mass spectrometry or MS/MS data with a database, and
providing said collated data for structural analysis to identify the expressed protein and/or peptide.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 10/582,090Filed 13 Dec 2004
Priority: JP 2003-41281011 Dec 2003
Published: US 2008/0280316 A113 Nov 2008
Granted: 5 Aug 2014
Term ends: Not estimatedThe source records don’t support an estimate.

Classification14 codes

G01N 33/582
Involving labelled substances: with fluorescent label
C07D 285/10
Not condensed with other rings: 1,2,5-Thiadiazoles; Hydrogenated 1,2,5-thiadiazoles
C07K 1/13
General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length:…
C07K 1/36
Extraction; Separation; Purification: by a combination of two or more processes of different types